Project Summary Immune checkpoint blockade (ICB) represents a paradigm shift in cancer treatment. ICB is effective against several cancer types, including particularly aggressive cancers such as metastatic melanoma. Remarkably, some patients treated with ICB experience durable responses previously unseen in metastatic disease, lasting upwards of 10 years. However, the majority of patients do not respond to ICB or relapse after therapy necessitating a deeper understanding of the molecular determinants of ICB response. Through the proposed work, I will characterize the role of the T cell repertoire in determining response to immune checkpoint blockade. Clinical studies have established a positive correlation between tumor-specific ?neo-antigen? (Ag) burden and response to ICB therapy, suggesting that ICB response is limited by the ability of T cells to recognize tumors. However, these studies also suggest that patients with low neo-Ag load will not benefit from ICB therapy, necessitating sensitizing therapies. I have found that treatment with a receptor activator of nuclear factor kappa-B ligand (RANKL) blocking antibody reduces tumor burden in models with limited neo-Ag repertoires; furthermore, RANKL blockade sensitizes these tumors to ICB therapy, resulting in complete remissions. My collaborator has shown that RANKL blockade inhibits central tolerance by depleting AIRE-medullary epithelial cells (mTECs), leading to the release of self-reactive T cells usually depleted in the thymus. Based on these observations, I hypothesize that temporary inhibition of central tolerance resulting from the depletion of AIRE-expressing mTECs mobilizes self-reactive T cells in the thymus to promote effective T cell-dependent responses against tumors that are otherwise ICB-resistant and/or harbor few somatic mutations (SM-low). In Aim 1, I will determine if the depletion of AIRE-expressing mTECs caused by RANKL blockade is responsible for the resulting decrease in tumor burden and restored sensitivity to ICB. To do this, I will use a genetic model of AIRE depletion to determine if temporary ablation of AIRE-expressing mTECs is sufficient to recapitulate the anti-tumor effects of RANKL blockade with and without ICB therapy against ICB-resistant and SM-low tumors. I will then characterize the effect of mTEC depletion on the activation and function of the T cells using multi-parameter flow cytometry and deplete T cells in vivo to establish that the anti-tumor effects observed after mTEC depletion are T cell-mediated. In addition, I will use previously characterized tetramers4,5 and peptides6 to determine whether depletion of mTECs mobilizes T cells reactive to known self-Ag epitopes. In Aim 2, I will characterize the fate and function of T cells that are generated during the period of mTEC depletion after RANKL blockade. Using Rag2-GFP mice, I will isolate GFP+ recent thymic emigrants (RTEs), and adoptively transfer them to congenic recipients to establish the extent to which the transferred RTEs participate in tumor responses. I will concurrently determine if these transferred RTEs are enriched for self- reactive T cells. In addition, I will use bioinformatic approaches to test if either self-Ags, neo-Ags, or both are depleted as a result of RANKL blockade with and without ICB therapy. If these Ags are depleted, I will functionally validate their depletion in future studies to establish novel antigen prediction pipelines. These studies will provide key insight into the role of the T cell repertoire in cancer immunotherapy. Furthermore, RANKL blockade therapy (Denosumab) is already clinically approved; therefore, I expect that this work will directly contribute to improved patient outcomes.